The invention relates to the field of biotechnology. In particular, the invention relates to a novel polypeptide, human calcium binding protein 42, and a polynucleotide sequence encoding said polypeptide. The invention also relates to the method for the preparation and use of said polynucleotide and polypeptide.
Calcium functions as a medium in many biological activities in cell. These biological activities include gene regulation, cell cycle regulation, signal transduction, release of brain neurotransmitter and decomposition of glycogen involved in muscle contraction and so on. These functions are activated by calcium that are released by stimulation of extracellular signals. Calcium binds with various calcium binding protein to mediate further signals which activate other molecules and effect some specific physiological functions.
Calcium binding protein family is a very large protein super family with a calcium binding motif named xe2x80x9cEF-hand,xe2x80x9d which is the binding site of the protein and calcium. Many calcium binding proteins have several EF-hand motifs. Approximately 250 calcium binding proteins of this type have been identified.
Calmodulin (CaM) is the most widely distributed calcium regulatory protein. Calmodulin contains four EF-hand motifs. When they bind with calcium, corresponding conformation change will occur. The activation of calmodulin will cause it to bind with other target proteins and change their activities. The main target protein of calmodulin is calmodulin-dependent protein kinase, which is involved in the regulation of smooth muscle contraction, glycogen metabolism and neurotransmitter release, while calciuneurin, another calmodulin-binding protein, is involved in brain synaptic transmission. Calmodulin plays an important role in these physiological processes. Its abnormal expression often adversely affects these normal physiological processes and leads to corresponding diseases, such as those related to abnormal release of neurotransmitter, including nervous system development disorder, nervous system degenerative diseases, neuromuscular disorder, neurocutaneous syndrome and so on.
Calcyphosine is another calcium binding protein that is co-regulated by calcium binding and phosphorylation. The dog calcyphosine P24 is a calcium binding protein with 3 EF-hand motifs and phosphorylated by cAMP-dependent protein kinase (Lefort, A., et al. 1989, EMBO, 8: 111-116). P24 is expressed in various secretory tissues such as salivary gland, lung and brain. This shows that P24 plays an important role in ion transport. In 1993, Nemoto Y. et al cloned a similar calcium binding phosphorylating protein R2D2 that regulates the signal transduction in olfactory nerve cells from hare. R2D2 has 3 EF-hand motifs and is phophorylated by cAMP-dependent protein kinase and CaM kinase (Nemoto Y. et al., 1993, J. Cell Biol., 123: 963-76). In the same year, Yasuo Nemoto et al. cloned an R2D2 antigen protein of 189 amino acid from hare. This protein has 3 EF-hand motifs, a Ca2+/calmodulin-dependent protein kinase II phosphorylation site and a cAMP-dependent kinase phosphorylation site. It is the first calcium binding protein found in olfactory sensory neurons that regulate olfactory signal transduction (Yasuo Nemoto, Jun Ikeda et al., 1993, J. Cell Biol., 123: 963-976). It is involved in the olfactory function, and its abnormal expression will lead to diseases related to olfactory organs.
The human calcium binding protein 42 of the present invention shares 32% identity and 55% similarity with the hare R2D2 antigen. They possess similar structural characteristics, that is, both of them have calcium binding EF-hand motif, Ca2+/calmodulin-dependent protein kinaseII phosphorylation site and cAMP-dependent protein kinase phosphorylation site. So both of them are calcium binding protein.
One objective of the invention is to provide an isolated novel polypeptide, i.e., a human calcium binding protein 42, and fragments, analogues and derivatives thereof.
Another objective of the invention is to provide a polynucleotide encoding said polypeptide.
Another objective of the invention is to provide a recombinant vector containing a polynucleotide encoding a human calcium binding protein 42.
Another objective of the invention is to provide a genetically engineered host cell containing a polynucleotide encoding a human calcium binding protein 42.
Another objective of the invention is to provide a method for producing a human calcium binding protein 42.
Another objective of the invention is to provide an antibody against a human calcium binding protein 42 of the invention.
Another objective of the invention is to provide mimetics, antagonists, agonists, and inhibitors for the polypeptide of the human calcium binding protein 42.
Another objective of the invention is to provide a method for the diagnosis and treatment of the diseases associated with an abnormality of human calcium binding protein 42.
The present invention relates to an isolated polypeptide, which is originated from human, and comprises a polypeptide having the amino acid sequence of SEQ ID NO: 2, or its conservative variants, or its active fragments, or its active derivatives and its analogues. Preferably, the polypeptide has the amino acid sequence of SEQ ID NO: 2.
The present invention also relates to an isolated polynucleotide, comprising a nucleotide sequence or its variant selected from the group consisting of (a) the polynucleotide encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 2; and (b) a polynucleotide complementary to the polynucleotide (a); (c) a polynucleotide that shares at least 70% homology to the polynucleotide (a) or (b). Preferably, said nucleotide sequence is selected from the group consisting of (a) the sequence of position 1022-2170 in SEQ ID NO: 1; and (b) the sequence of position 1-3829 in SEQ ID NO: 1.
The invention also includes: a vector containing a polynucleotide of said invention, especially an expression vector; a host cell genetically engineered with the vector via transformation, transduction or transfection; a method for the production of said inventive polypeptide through the process of host cell cultivation and expression product harvest.
The invention also relates to an antibody which specifically binds to the inventive polypeptide.
The invention also relates to a method for selecting compounds which could simulate, activate, antagonize, or inhibit the activity of the inventive polypeptide and the compounds obtained by the method.
The invention also relates to a method for in vitro diagnosis method of the diseases or disease susceptibility related with the abnormal expression of the inventive polypeptide. The method involves the detection of mutation in the polypeptide or its encoding polynucleotide sequence, or the determination of its quantity and/or biological activity in biological samples.
The invention also relates to pharmaceutical compositions which comprises the inventive polypeptide, its analogues, mimetics, agonists, antagonists, inhibitors, and a pharmaceutically acceptable carrier.
The invention also relates to applications of the inventive polypeptide and/or its polynucleotide for drug development to treat cancers, developmental diseases, immune diseases, or other diseases caused by abnormal expression of the inventive polypeptide.
Other aspects of the invention are apparent to the skilled in the art in view of the disclosure set forth hereinbelow.
The terms used in this specification and claims have the following meanings, unless otherwise noted.
xe2x80x9cNucleotide sequencexe2x80x9d refers to oligonucleotide, nucleotide, or polynucleotide and parts of polynucleotide. It also refers to genomic or synthetic DNA or RNA, which could be single stranded or double stranded, and could represent the sense strand or the antisense strand. Similarly, the term xe2x80x9camino acid sequencexe2x80x9d refers to oligopeptide, peptide, polypeptide, or protein sequence and parts of proteins. When the xe2x80x9camino acid sequencexe2x80x9d in the invention is related to the sequence of a natural protein, the amino acid sequence of said xe2x80x9cpeptidexe2x80x9d or xe2x80x9cproteinxe2x80x9d will not be limited to be identical to the sequence of that natural protein.
xe2x80x9cvariantxe2x80x9d of a protein or polynucleotide refers to the amino acid sequence or nucleotide sequence, respectively with one or more amino acids or one or more nucleotides changed. Such changes include deletion, insertion, and/or substitution of amino acids in the amino acid sequence, or of nucleotides in the polynucleotide sequence. These changes could be conservative and the substituted amino acid has similar structural or chemical characteristics as the original one, such as the substitution of Ile with Leu. Changes also could be not conservative, such as the substitution of Ala with Trp.
xe2x80x9cDeletionxe2x80x9d refers to the deletion of one or several amino acids in the amino acid sequence, or of one or several nucleotides in the nucleotide sequence.
xe2x80x9cInsertionxe2x80x9d or xe2x80x9cadditionxe2x80x9d refers to the addition of one or several amino acids in the amino acid sequence, or of one or several nucleotides in the nucleotide sequence, comparing to the natural molecule. xe2x80x9cSubstitutionxe2x80x9d refers to the change of one or several amino acids, or of one or several nucleotides, into different ones without changing number of the residues.
xe2x80x9cBiological activityxe2x80x9d refers to structural, regulatory or biochemical characteristics of a natural molecule. Similarly, the term xe2x80x9cimmungenecityxe2x80x9d refers to the ability of natural, recombinant, or synthetic proteins to inducing a specific immunological reaction, or of binding specific antibody in appropriate kind of animal or cell.
xe2x80x9cAgonistxe2x80x9d refers to molecules which regulate, but generally enhance the activity of the inventive polypeptide by binding and changing it. Agonists include proteins, nucleotides, carbohydrates or any other molecules which could bind the inventive polypeptide.
xe2x80x9cAntagonistxe2x80x9d or xe2x80x9cinhibitorxe2x80x9d refers to molecules which inhibit or down regulate the biological activity or immunogenecity the inventive polypeptide via binding to it. Antagonists or inhibitors include proteins, nucleotides, carbohydrates or any other molecules which bind to the inventive polypeptide.
xe2x80x9cRegulationxe2x80x9d refers to changes in function of the inventive polypeptide, including up-regulation or down-regulation of the protein activity, changes in binding specificity, changes of any other biological characteristics, functional or immune characteristics.
xe2x80x9cSubstantially purexe2x80x9d refers to the condition of substantially free of other naturally related proteins, lipids, saccharides, or other substances. One of ordinary skill in the art can purify the inventive polypeptide by standard protein purification techniques. Substantially pure polypeptide of the invention produces a single main band in a denaturing polyacrylamide gel. The purity of a polypeptide may also be analyzed by amino acid sequence analysis.
xe2x80x9cComplementaryxe2x80x9d or xe2x80x9ccomplementationxe2x80x9d refers to the binding of polynucleotides by base pairing under the condition of approximate ion conditions and temperature. For instance, the sequence xe2x80x9cC-T-G-Axe2x80x9d could bind its complementary sequence xe2x80x9cG-A-C-T.xe2x80x9d The complementation between two single strand molecules could be partial or complete. Homology or sequence similarity between two single strands obviously influences the efficiency and strength of the formed hybrid.
xe2x80x9cHomologyxe2x80x9d refers to the complementary degree, which may be partially or completely homologous. xe2x80x9cPartial homologyxe2x80x9d refers to a sequence being partially complementary to a target nucleotide. The sequence could at least partially inhibit the hybridization between a completely complementary sequence and the target nucleotide. Inhibition of hybridization could be assayed by hybridization (Southern blot or Northern blot) under less stringent conditions. Substantially complementary sequence or hybrid probe could compete with the completely complementary sequence and inhibit its hybridization with the target sequence under less stringent conditions. This doesn""t mean that nonspecific binding is allowed under a less stringent condition, because specific or selective reaction is still required.
xe2x80x9cSequence Identityxe2x80x9d refers to the percentage of sequence identity or similarity when two or several amino acid or nucleotide sequences are compared. Sequence identity may be determined by computer programs such as MEGALIGN (Lasergene Software Package, DNASTAR, Inc., Madison Wis.). MEGALIGN can compare two or several sequences using different methodologies such as the Cluster method (Higgins, D. G. and P. M. Sharp (1988) Gene 73: 237-244). Cluster method examines the distance between all pairs and arrange the sequences into clusters. Then the clusters are partitioned by pair or group. The sequence identity between two amino acid sequences such as sequence A and B can be calculated by the following equation:                                           Number            ⁢                          xe2x80x83                        ⁢            of            ⁢                          xe2x80x83                        ⁢            paired            ⁢                          xe2x80x83                        ⁢            identical            ⁢                          xe2x80x83                        ⁢            residues                                                            between            ⁢                          xe2x80x83                        ⁢            sequences            ⁢                          xe2x80x83                        ⁢            A            ⁢                          xe2x80x83                        ⁢            and            ⁢                          xe2x80x83                        ⁢            B                                                                                  Residue              ⁢                              xe2x80x83                            ⁢              number              ⁢                              xe2x80x83                            ⁢              of              ⁢                              xe2x80x83                            ⁢              sequence              ⁢                              xe2x80x83                            ⁢              A                        -                                                                          number              ⁢                              xe2x80x83                            ⁢              of              ⁢                              xe2x80x83                            ⁢              gap              ⁢                              xe2x80x83                            ⁢              residues              ⁢                              xe2x80x83                            ⁢              in              ⁢                              xe2x80x83                            ⁢              sequence              ⁢                              xe2x80x83                            ⁢              A                        -                                                            number            ⁢                          xe2x80x83                        ⁢            of            ⁢                          xe2x80x83                        ⁢            gap            ⁢                          xe2x80x83                        ⁢            residue            ⁢                          xe2x80x83                        ⁢            in            ⁢                          xe2x80x83                        ⁢            sequence            ⁢                          xe2x80x83                        ⁢            B                                xc3x97  100
Sequence identity between nucleotide sequences can also be determined by Cluster method or other well-known methods in the art such as the Jotun Hein method (Hein J., (1990) Methods in Enzymology 183: 625-645)
xe2x80x9cSimilarityxe2x80x9d refers to the degree of identity or conservative substitution degree of amino acid residues in corresponding sites of the amino acid sequences when compared to each other. Amino acids for conservative substitution are: negative charged amino acids including Asp and Glu; positive charged amino acids including Leu, Ile and Val; Gly and Ala; Asn and Gln; Ser and Thr; Phe and Tyr.
xe2x80x9cAntisensexe2x80x9d refers to the nucleotide sequences complementary to a specific DNA or RNA sequence. xe2x80x9cAntisense strandxe2x80x9d refers to the nucleotide strand complementary to the xe2x80x9csense strand.xe2x80x9d
xe2x80x9cDerivativexe2x80x9d refers to the inventive polypeptide or the chemically modified nucleotide encoding it. This kind of modified chemical can be derived from replacement of the hydrogen atom with Alkyl, Acyl, or Amino. The nucleotide derivative can encode peptide retaining the major biological characteristics of the natural molecule.
xe2x80x9cAntibodyxe2x80x9d refers to the intact antibody or its fragments such as Fa, F(abxe2x80x2)2 and Fv, and it can specifically bind to antigenic epitopes of the inventive polypeptide.
xe2x80x9cHumanized antibodyxe2x80x9d refers to an antibody which has its amino acid sequence in non-antigen binding region replaced to mimic human antibody and still retain the original binding activity.
The term xe2x80x9cisolatedxe2x80x9d refers to the removal of a material out of its original environment (for instance, if it""s naturally produced, original environment refers to its natural environment). For example, a naturally produced polynucleotide or a polypeptide in its original host organism means it has not been xe2x80x9cisolated,xe2x80x9d while the separation of the polynucleotide or a polypeptide from its coexisting materials in natural system means it was xe2x80x9cisolated.xe2x80x9d This polynucleotide may be a part of a vector, or a part of a compound. Since the vector or compound is not part of its natural environment, the polynucleotide or peptide is still xe2x80x9cisolated.xe2x80x9d
As used herein, the term xe2x80x9cisolatedxe2x80x9d refers to a substance which has been isolated from the original environment. For naturally occurring substance, the original environment is the natural environment. For example, the polynucleotide and polypeptide in a naturally occurring state in the viable cells are not isolated or purified. However, if the same polynucleotide and polypeptide have been isolated from other components naturally accompanying them, they are isolated or purified.
As used herein, xe2x80x9cisolated human calcium binding protein 42,xe2x80x9d means that human calcium binding protein 42 does not essentially contain other proteins, lipids, carbohydrate or any other substances associated therewith in nature. The skilled in the art can purify human calcium binding protein 42, by standard protein purification techniques. The purified polypeptide forms a single main band on a non-reducing PAGE gel. The purity of human calcium binding protein 42 can also be analyzed by amino acid sequence analysis.
The invention provides a novel polypeptidexe2x80x94human calcium binding protein 42, which comprises the amino acid sequence shown in SEQ ID NO: 2. The polypeptide of the invention may be a recombinant polypeptide, natural polypeptide, or synthetic polypeptide, preferably a recombinant polypeptide. The polypeptide of the invention may be a purified natural product or a chemically synthetic product. Alternatively, it may be produced from prokaryotic or eukaryotic hosts, such as bacterial, yeast, higher plant, insect, and mammal cells, using recombinant techniques. Depending on the host used in the protocol of recombinant production, the polypeptide of the invention may be glycosylated or non-glycosylated. The polypeptide of the invention may or may not comprise the starting Met residue.
The invention further comprises fragments, derivatives and analogues of human calcium binding protein 42. As used in the invention, the terms xe2x80x9cfragment,xe2x80x9d xe2x80x9cderivativexe2x80x9d and xe2x80x9canaloguexe2x80x9d mean the polypeptide that essentially retains the same biological functions or activity of human calcium binding protein 42 of the invention. The fragment, derivative or analogue of the polypeptide of the invention may be (i) one in which one or more of the amino acid residues are substituted with a conserved or non-conserved amino acid residue (preferably a conserved amino acid residue) and such substituted amino acid residue may or may not be one encoded by the genetic code; or (ii) one in which one or more of the amino acid residues are substituted with other residues, including a substituent group; or (iii) one in which the mature polypeptide is fused with another compound, such as a compound to increase the half-life of the polypeptide (for example, polyethylene glycol); or (iv) one in which additional amino acids are fused to the mature polypeptide, such as a leader or secretory sequence or a sequence which is employed for purification of the mature polypeptide or a proprotein sequence. Such fragments, derivatives and analogs are deemed to be within the scope of the skilled in the art from the teachings herein.
The invention provides an isolated nucleic acid or polynucleotide which comprises the polynucleotide encoding an amino acid sequence of SEQ ID NO: 2. The polynucleotide sequence of the invention includes the nucleotide sequence of SEQ ID NO: 1. The polynucleotide of the invention was identified in a human embryonic brain cDNA library. Preferably, it comprises a full-length polynucleotide sequence of 3829 bp, whose ORF (1022-2170) encodes 382 amino acids. Based on amino acid homology comparison, it is found that the encoded polypeptide is 32% homologous to hare R2D2 antigen. This novel human calcium binding protein 42 has similar structures and biological functions to those of the hare R2D2 antigen.
The polynucleotide according to the invention may be in the forms of DNA or RNA. The forms of DNA include cDNA, genomic DNA, and synthetic DNA, etc., in single stranded or double stranded form. DNA may be an encoding strand or a non-encoding strand. The coding sequence for mature polypeptide may be identical to the coding sequence shown in SEQ ID NO: 1, or is a degenerate sequence. As used herein, the term xe2x80x9cdegenerate sequencexe2x80x9d means a sequence which encodes a protein or peptide comprising a sequence of SEQ ID NO: 2 and which has a nucleotide sequence different from the sequence of coding region in SEQ ID NO: 1.
The polynucleotide encoding the mature polypeptide of SEQ ID NO: 2 includes those encoding only the mature polypeptide, those encoding mature polypeptide plus various additional coding sequence, the coding sequence for mature polypeptide (and optional additional encoding sequence) plus the non-coding sequence.
The term xe2x80x9cpolynucleotide encoding the polypeptidexe2x80x9d includes polynucleotides encoding said polypeptide and polynucleotides comprising additional coding and/or non-coding sequences.
The invention further relates to variants of the above polynucleotides which encode a polypeptide having the same amino acid sequence of invention, or a fragment, analogue and derivative of said polypeptide. The variant of the polynucleotide may be a naturally occurring allelic variant or a non-naturally occurring variant. Such nucleotide variants include substitution, deletion, and insertion variants. As known in the art, an allelic variant may have a substitution, deletion, and insertion of one or more nucleotides without substantially changing the functions of the encoded polypeptide.
The present invention further relates to polynucleotides, which hybridize to the hereinabove-described sequences, that is, there is at least 50% and preferably at least 70% identity between the sequences. The present invention particularly relates to polynucleotides, which hybridize to the polynucleotides of the invention under stringent conditions. As herein used, the term xe2x80x9cstringent conditionsxe2x80x9d means the following conditions: (1) hybridization and washing under low ionic strength and high temperature, such as 0.2xc3x97SSC, 0.1% SDS, 60xc2x0 C.; or (2) hybridization after adding denaturants, such as 50% (v/v) formamide, 0.1% bovine serum/0.1% Ficoll, 42xc2x0 C.; or (3) hybridization only when the homology of two sequences at least 95%, preferably 97%. Further, the polynucleotides which hybridize to the hereinabove described polynucleotides encode a polypeptide which retains the same biological function and activity as the mature polypeptide of SEQ ID NO: 2.
The invention also relates to nucleic acid fragments hybridized with the hereinabove sequence. As used in the present invention, the length of the xe2x80x9cnucleic acid fragmentxe2x80x9d is at least more than 10 bp, preferably at least 20-30 bp, more preferably at least 50-60 bp, and most preferably at least 100 bp. The nucleic acid fragment can be used in amplification techniques of nucleic acid, such as PCR, so as to determine and/or isolate the polynucleotide encoding human calcium binding protein 42.
The polypeptide and polynucleotide of the invention are preferably in the isolated form, preferably purified to be homogenous.
According to the invention, the specific nucleic acid sequence encoding human calcium binding protein 42 can be obtained in various ways. For example, the polynucleotide is isolated by hybridization techniques well-known in the art, which include, but are not limited to 1) the hybridization between a probe and genomic or cDNA library so as to select a homologous polynucleotide sequence, and 2) antibody screening of expression library so as to obtain polynucleotide fragments encoding polypeptides having common structural features.
According to the invention, DNA fragment sequences may further be obtained by the following methods: 1) isolating double-stranded DNA sequence from genomic DNA; and 2) chemical synthesis of DNA sequence so as to obtain the double-stranded DNA.
Among the above methods, the isolation of genomic DNA is least frequently used. A commonly used method is the direct chemical synthesis of DNA sequence. A more frequently used method is the isolation of cDNA sequence. Standard methods for isolating the cDNA of interest is to isolate mRNA from donor cells that highly express said gene followed by reverse transcription of mRNA to form plasmid or phage cDNA library. There are many established techniques for extracting mRNA and the kits are commercially available (e.g. Qiagene). Conventional method can be used to construct cDNA library (Sambrook, et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory. New York, 1989). The cDNA libraries are also commercially available. For example, Clontech Ltd. has various cDNA libraries. When PCR is further used, even an extremely small amount of expression products can be cloned.
Numerous well-known methods can be used for screening for the polynucleotide of the invention from cDNA library. These methods include, but are not limited to, (1) DNA-DNA or DNA-RNA hybridization; (2) the appearance or loss of the function of the marker-gene; (3) the determination of the level of human calcium binding protein 42 transcripts; (4) the determination of protein product of gene expression by immunology methods or the biological activity assays. The above methods can be used alone or in combination.
In method (1), the probe used in the hybridization could be homologous to any portion of polynucleotide of invention. The length of probe is typically at least 10 nucleotides, preferably at least 30 nucleotides, more preferably at least 50 nucleotides, and most preferably at least 100 nucleotides. Furthermore, the length of the probe is usually less than 2000 nucleotides, preferably less than 1000 nucleotides. The probe usually is the DNA sequence chemically synthesized on the basis of the sequence information. Of course, the gene of the invention itself or its fragment can be used as a probe. The labels for DNA probe include, e.g., radioactive isotopes, fluoresceins or enzymes such as alkaline phosphatase.
In method (4), the detection of the protein products expressed by human calcium binding protein 42 gene can be carried out by immunology methods, such as Western blotting, radioimmunoassay, and ELISA.
The method of amplification of DNA/RNA by PCR (Saiki, et al. Science 1985; 230:1350-1354) is preferably used to obtain the polynucleotide of the invention. Especially when it is difficult to obtain the full-length cDNA, the method of RACE (RACE-cDNA terminate rapid amplification) is preferably used. The primers used in PCR can be selected according to the polynucleotide sequence information of the invention disclosed herein, and can be synthesized by conventional methods. The amplified DNA/RNA fragments can be isolated and purified by conventional methods such as gel electrophoresis.
Sequencing of polynucleotide sequence of the gene of the invention or its various DNA fragments can be carried out by the conventional dideoxy sequencing method (Sanger et al. PNAS, 1977, 74: 5463-5467). Sequencing of polynucleotide sequence can also be carried out using the commercially available sequencing kits. In order to obtain the full-length cDNA sequence, it is necessary to repeat the sequencing process. Sometimes, it is needed to sequence the cDNA of several clones to obtain the full-length cDNA sequence.
The invention further relates to a vector comprising the polynucleotide of the invention, a genetically engineered host cell transformed with the vector of the invention or directly with the sequence encoding human calcium binding protein 42, and a method for producing the polypeptide of the invention by recombinant techniques.
In the present invention, the polynucleotide sequences encoding human calcium binding protein 42 may be inserted into a vector to form a recombinant vector containing the polynucleotide of the invention. The term xe2x80x9cvectorxe2x80x9d refers to a bacterial plasmid, bacteriophage, yeast plasmid, plant virus or mammalian virus such as adenovirus, retrovirus or any other vehicle known in the art. Vectors suitable for use in the present invention include, but are not limited to the T7-based expression vector for expression in bacteria (Rosenberg, et al., Gene, 56:125, 1987), the pMSXND expression vector for expression in mammalian cells (Lee and Nathans, J Biol. Chem., 263:3521, 1988) and baculovirus-derived vectors for expression in insect cells. Any plasmid or vector can be used to construct the recombinant expression vector as long as it can replicate and is stable in the host. One important feature of an expression vector is that the expression vector typically contains an origin of replication, a promoter, a marker gene as well as translation regulatory components.
Methods known in the art can be used to construct an expression vector containing the DNA sequence of human calcium binding protein 42 and appropriate transcription/translation regulatory components. These methods include in vitro recombinant DNA technique, DNA synthesis technique, in vivo recombinant technique and so on (Sambroook, et al. Molecular Cloning, a Laboratory Manual, Cold Spring Harbor Laboratory, New York, 1989). The DNA sequence is operatively linked to a proper promoter in an expression vector to direct the synthesis of mRNA. Exemplary promoters are lac or trp promoter of E. coli; PL promoter of xcex phage; eukaryotic promoters including CMV immediate early promoter, HSV thymidine kinase promoter, early and late SV40 promoter, LTRs of retrovirus, and other known promoters which control gene expression in the prokaryotic cells, eukaryotic cells or viruses. The expression vector may further comprise a ribosome binding site for initiating translation, transcription terminator and the like. Transcription in higher eukaryotes is increased by inserting an enhancer sequence into the vector. Enhancers are cis-acting elements of DNA, usually about from 10 to 300 bp in length that act on a promoter to increase gene transcription level. Examples include the SV40 enhancer on the late side of the replication origin 100 to 270 bp, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers.
Further, the expression vector preferably comprises one or more selective marker genes to provide a phenotype for the selection of the transformed host cells, e. g. , the dehydrofolate reductase, neomycin resistance gene and GFP (green flurencent protein) for eukaryotic cells, as well as tetracycline or ampicillin resistance gene for E. coli. 
An ordinarily skilled in the art know clearly how to select appropriate vectors, transcriptional regulatory elements, e.g., promoters, enhancers, and selective marker genes.
According to the invention, polynucleotide encoding human calcium binding protein 42 or recombinant vector containing said polynucleotide can be transformed or transfected into host cells to construct genetically engineered host cells containing said polynucleotide or said recombinant vector. The term xe2x80x9chost cellxe2x80x9d means prokaryote, such as bacteria; or primary eukaryote, such as yeast; or higher eukaryotic, such as mammalian cells. Representative examples are bacterial cells, such as E. coli, Streptomyces, Salmonella typhimurium; fungal cells, such as yeast; plant cells; insect cells such as Drosophila S2 or Sf9; animal cells such as CHO, COS or Bowes melanoma.
Transformation of a host cell with the DNA sequence of invention or a recombinant vector containing said DNA sequence may be carried out by conventional techniques as are well known to those skilled in the art. When the host is prokaryotic, such as E. coli, competent cells, which are capable of DNA uptake, can be prepared from cells harvested after exponential growth phase and subsequently treated by the CaCl2 method using procedures well known in the art. Alternatively, MgCl2 can be used. Transformation can also be carried out by electroporation, if desired. When the host is an eukaryote, transfection methods as well as calcium phosphate precipitation may be used. Conventional mechanical procedures such as micro-injection, electroporation, or liposome-mediated transfection may also be used.
The recombinant human calcium binding protein 42 can be expressed or produced by the conventional recombinant DNA technology (Science, 1984; 224:1431), using the polynucleotide sequence of the invention. The steps generally include:
(1) transfecting or transforming the appropriate host cells with the polynucleotide (or variant) encoding human calcium binding protein 42 of the invention or the recombinant expression vector containing said polynucleotide;
(2) culturing the host cells in an appropriate medium; and
(3) isolating or purifying the protein from the medium or cells.
In Step (2) above, depending on the host cells used, the medium for cultivation can be selected from various conventional mediums. The host cells are cultured under a condition suitable for its growth until the host cells grow to an appropriate cell density. Then, the selected promoter is induced by appropriate means (e.g., temperature shift or chemical induction) and cells are cultured for an additional period.
In Step (3), the recombinant polypeptide may be included in the cells, or expressed on the cell membrane, or secreted out of the cell. If desired, physical, chemical and other properties can be utilized in various isolation methods to isolate and purify the recombinant protein. These methods are well-known to those skilled in the art and include, but are not limited to conventional renaturation treatment, treatment by a protein precipitant (such as salt precipitation), centrifugation, cell lysis by osmosis, sonication, supercentrifugation, molecular sieve chromatography or gel chromatography, adsorption chromatography, ion exchange chromatography, HPLC, and any other liquid chromatography, and a combination thereof.